Cocaine is a prominent cofactor in the epidemiology of HIV, associated with risky behavior that increases exposure to virus and undermines adherence to treatment. But independent of behavioral factors, cocaine appears to increase HIV replication and accelerate progression to AIDS. We hypothesize this occurs because cocaine facilitates HIV subversion of key host defenses. How does HIV itself subvert immune defenses? One pathway involves dendritic cells (DCs) that normally capture pathogens and degrade them in lysosomes. HIV can bypass this pathway by complexing with DC-SIGN and cytoskeletal proteins, trafficking to endosomes rather than lysosomes, so it is not degraded. Endosome-associated virus is then richly presented by DCs to CD4+ T-cells via the immune synapse. HIV can also subvert an intact lymphatic endothelial (LE) barrier through altering novel Robo receptors which normally signal to stabilize the cytoskeleton. Robo dysregulation increases endothelial permeability, which would promote virus dissemination into the bloodstream. Similar HIV effects may occur in high endothelial venules (HEVs), the gatekeepers for immune cell trafficking in lymph nodes. Studies show cocaine can specifically alter cytoskeletal and signaling pathways in DCs and endothelium that would work to the benefit of HIV. The overall objective of this proposal is to gain a deeper understanding of how HIV and cocaine partner on a molecular level to subvert host defenses. We will model their effects both in vitro and in vivo in the BLT humanized mouse, and explore how targeting novel Robo receptors may oppose the deleterious changes triggered by cocaine that enhance HIV pathobiology. Our initial studies support this strategy: Robo signaling protected LE from HIV hyperpermeability and in DCs inhibited virus transmission to T-cells. Specific points of innovation include: (1) focus on alterations of the cytoskeletal apparatus as a unique way HIV and cocaine may disarm immune responses; (2) utilize purified populations of LEs and HEVs, whose roles in limiting HIV are not yet well characterized; (3) exploit the newly identified ligands and receptors of the Slit/Robo family in antagonizing cocaine-facilitated HIV subversion of immune defense; (4) apply cutting-edge methodology in the in vivo HIV-BLT mouse model to translate in vitro observations. The specific aims are: (1) further characterize molecular effects of HIV and cocaine in DCs that enhance virus transmission to T-cells; (2) characterize molecular changes in LE and HEV barriers due to HIV and cocaine, and further assess protective effects of Robo signaling; (3) model in vitro DC and T-cell interactions with LE and HEV in the presence of HIV and cocaine; (4) assess in vivo in the BLT humanized mouse how Robo signaling may antagonize cocaine-enhanced HIV pathobiology. By generating this new knowledge, we hope to provide the foundation for innovative strategies to prevent or contain HIV infection in users of cocaine.